GPCRs on cell membranes participate in a variety of cell signaling pathways and are one of the most popular targets for new therapeutics. Currently around 30% of clinically prescribed drugs act on GPCR family members. (see “Application Notes: Time-resolved fluorescence based GTP binding assay for G-Protein coupled receptors,” PerkinElmer Life Sciences, www.perkinelmer.com/lifesciences). Moreover, GPCRs are associated with almost every major therapeutic category or disease class, including pain, asthma, inflammation, obesity, cancer, as well as cardiovascular, metabolic, gastrointestinal and central nervous system diseases. The tremendous significance of drugs targeting GPCRs lies in the physiological roles of GPCRs as cell-surface receptors responsible for transducing exogenous signals into intracellular response(s). (see Haga, T., and Berstein, G., G-Protein-Coupled Receptors, CRC Press, Boca Raton, Fla., 1999.) Signaling through these receptors regulates a wide variety of physiological processes, such as neurotransmission, chemotaxis, inflammation, cell proliferation, cardiac and smooth muscle contractility, as well as visual and chemosensory perception. In addition to the role that normal receptors play in modulating physiological processes, GPCR mutations that result in both gain and loss of function are associated with certain human diseases. For example, GPCR polymorphisms have been linked with hypertension, idiopathic cardiomyopathy (endothelin A receptor), autosomal dominant hypocalcemia and familial hypocalciuric hypercalcemia (calcium-sensing receptor), follicular maturation arrest and suppression of spermatogenesis (follicle-stimulating hormone receptor), and bronchodilator desensitization and nocturnal asthma (β2-adrenoceptors).
In the human genome there are about 400-700 GPCRs of therapeutic relevance; of these GPCRs, ligands for about 200 have been discovered. (see Pierce, K. L. et al., “Seven-Transmembrane Receptors.” Nat. Rev. Mol. Cell Biol. 2002, v. 3, 639-650.) Although there is very little conservation at the amino acid level among GPCR sequences, all GPCRs share certain structural and mechanistic features. Typically, GPCRs are formed of seven-helical trans-membrane-spanning domains (each approximately 20-30 amino acids in length) joined by intra- and extra-cellular loops. The spatial organization of these trans-membrane regions, the extra-cellular N-terminus and the extracellular loops, form the binding sites or testing targets for extra-cellular ligands. The intracellular loops and carboxyl-terminus form the sites of interaction with signal-transducing heterotrimeric G-proteins and other regulatory proteins, such as receptor kinases and arrestins. A wide variety of ligand species, including biogenic amines, peptides and proteins, lipids, nucleotides, excitatory amino acids and ions, small chemical compounds, etc., can activate GPCRs.
Functionally, the GPCR participated cell signaling pathway (signal transduction) begins with the binding of an extracellular substance (ion, small molecule, protein) to an extracellular domain of the GPCR. Binding of the substance to an extracellular domain of the transmembrane protein causes the protein to change from an inactive form to an active form. Once activated, the protein stimulates catalytic activity, or some similar such response, that generates a cytosolic signal (which is sometimes in the form of one or more secondary messenger substances in the cytoplasm). There are two major types of such signal transductions in mammalian cells: (i) the transmembrane protein may have a protein kinase activity in its cytosolic domain, the activity of which is activated when the extracellular substance binds to the transmembrane protein (the kinase then phosphorylates its own cytoplasmic domain, which enables the transmembrane protein to associate and activate another protein, which in turn acts on other proteins and substances within the cell cytoplasm); and (ii) the transmembrane protein may interact with a G protein that is associated with the membrane, which causes the GDP (guanine diphosphate) bound to the G protein to be replaced by GTP, resulting in dissociation of the G protein into monomer and dimmer fragments, one or both of which, in turn, acts upon a target protein (also often associated with the membrane, requiring it to then act upon yet another target protein, this one in the cytoplasm).
Given the importance of G-protein-coupled receptors as drug targets, a wide range of technologies have been developed that screen compounds against GPCRs. (e.g., Hemmila, I. A., and Hurskainen, P., “Novel Detection Strategies for Drug Discovery,” Drug Discov. Today 2002, 7, S152-S156.) Two types of assays that can be performed to screen GPCRs include GPCR binding assays and GPCR functional assays. GPCR binding assays screen extracellular molecules that can bind to the extracellular domain of the GPCRs, while GPCR functional assays screen the G-protein activation or deactivation by the extracellular molecules (agonist or antagonist) that bind to the GPCRs.
As is generally known, functional assays using GPCR microarrays are essential for investigating “orphan” GPCRs, some of which may turn out to be key drug targets. Orphan GPCRs are those without known ligands, which preclude the use of competition assays employing known labeled ligands. Functional assays can be both cell-based and biochemical in nature. Cell based assays include reporter gene assays, P-arrestin and GPCR-GFP translocation assays (i.e., receptor internalization and endosome formation). Methods for monitoring the activation of GPCRs by non-cell based assays are mostly limited to monitoring GTP-GDP exchange at the GPCR associated Gα protein using labeled GTP analogues (e.g., 35S-GTP γS or Europium-labeled GTP—“Eu-GTP”). These GPCR functional assays are typically performed in a homogeneous solution and use a radioactive labeling approach or Europium (Eu) label based time-delayed fluorescence. The receptor and the GTP analogue in a homogenous assay are mixed with or without a compound of interest and are in a solution over the duration of the assay. These assays are then subject to filtration using a filter microplate so that the labeled GTP can be removed by filtration, and only the bound GTP analog molecules can be quantified and the effect of the compound on the binding of GTP analog can be examined, which can be used to classify the action of the compound on the receptors (i.e., non-binder, or antagonist, or agonist, etc).
While Eu label based GPCR functional assays have significant advantages over traditional labeling assays, such as having no radioactivity risks, performing these functional assays in a homogeneous format limits their throughput characteristics. Porous substrate surfaces have been developed that allow GPCR functional assays to be performed in a micro-array format (see for example U.S. patent application Ser. No. 10/822,385 to Fang et al, the disclosure of which is incorporated in its entirety herein by this reference). These substrates comprise a 3-D structure that allows access to both sides of the membrane protein and enables the functional activation of the protein arrays. However, it has been discovered that traditional time-resolved fluorometers have difficulty measuring the array assay results on these substrates. Thus, it would be desirable to have a system for measuring the array assay results for porous substrate surfaces.